Abstract

Rationale

Human studies and mouse models have provided evidence for angiotensin II (AngII)-based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that AngII infusion induces aneurysmal pathology in the ascending aorta.

Objective

The aim of this study was to define the role of angiotensin II type 1a (AT1a) receptors and their cellular location in AngII-induced ascending aortic aneurysms (AAs).

Methods and Results

Male LDL receptor −/− mice were fed a saturated fat-enriched diet for 1 week prior to osmotic mini-pump implantation and infused with either saline or AngII (1,000 ng/kg/min) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT1a receptor deficiency ablated AngII-induced ascending AAs (P<0.001). To determine the role of AT1a receptors on leukocytes, LDL receptor −/− x AT1a receptor +/+ or −/− mice were irradiated and repopulated with bone marrow-derived cells isolated from either AT1a receptor +/+ or −/− mice. Deficiency of AT1a receptors in bone marrow-derived cells had no effect on AngII-induced ascending AAs. To determine the role of AT1a receptors on vascular wall cells, we developed AT1a receptor floxed mice with depletion on either smooth muscle (SMC) or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT1a receptor deletion in SMCs had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology.

Conclusions

AngII infusion promotes aneurysms in the ascending aorta via stimulation of AT1a receptors that are expressed on endothelial cells.

Keywords: ascending aneurysm, angiotensin II, AT1a receptor, Tek-cre

INTRODUCTION

Ascending aortic aneurysm (AA) is an asymptomatic expansion of this restricted region in which rupture has catastrophic consequences.1 It has now become apparent that ascending AAs have a significantly higher incidence than originally thought. Furthermore, studies and communities in which autopsies are routinely performed have demonstrated that the incidence of aortic aneurysms is increasing.2

Recent human and experimental studies have inferred a role for angiotensin II (AngII) in the development of ascending AAs.3–6 Ascending AAs can be generated in transgenic mice that express a common mutation of fibrillin-1 present in patients suffering from Marfan’s disease. There are also several other genes that have been associated with AAs.4 The aortic dilation that is localized to the ascending region is prevented by the administration of the AT1 receptor antagonist, losartan.5 Retrospective analysis of pharmacological treatments given to individuals suffering from Marfan’s disease demonstrated that administration of losartan attenuated dilation of the ascending aorta.3 Prospective evaluations of angiotensin receptor antagonists are currently being performed in populations afflicted with Marfan’s syndrome.7

AngII has diverse effects that could be implicated in aneurysm formation in the ascending aorta. AngII exerts its bioactive effects mainly via stimulation of AT1 receptors in most species, and the AT1a receptor subtype in rodents. This receptor subtype is ubiquitously present on the cell types involved in vascular pathology including endothelium, smooth muscle cells (SMCs), and macrophages.

We have demonstrated recently that AngII infusion into hypercholesterolemic mice promotes pronounced dilation that is localized to the ascending aorta.3 This region of lumen dilation also has concentric medial thickening that is most pronounced on the adventitia aspect of the aorta. In many mice infused with AngII, ulceration develops on the anterior aspect of ascending aortas. Using this model as an experimental paradigm of AAs, we determined the role of AT1a receptor expression and cellular localization on the development of AngII-induced ascending AAs.

Genotyping

Genotypes of mice were confirmed by PCR of genomic DNA.

Detection of AT1a receptor mRNA

Total RNA was isolated from liver, aortic SMCs and endothelial cells. cDNAs were transcribed using iScript cDNA synthesis kits and primers specific for AT1a receptor mRNA were used in real time PCR, with confirmation of amplicons by visualized detected on agarose gels.

Detection of β-galactosidase activity in tissues

Aortas were removed and β-galactosidase activity was detected by X-gal.

Detection of AT1a receptor protein

Several commercially available and newly developed antibodies were tested for specificity.

Systolic blood pressure measurements

Systolic blood pressure (SBP) was measured using the Kent Scientific or Visitech tail cuff machine as described previously.9

Serum measurements

Total serum cholesterol concentrations were determined as described previously.10 Serum MCP-1 concentration was measured by ELISA (R&D Systems).

Bone marrow transplantation

Bone marrow transplantation was performed as described previously.8,11

Aortic contractility studies

Aortic segments were isolated and subjected to KCl, 5-HT, and AngII incubations.

Measurements and pathohistology of ascending AAs

Ascending aortic aneurysms were measured by intimal area and elongation. Aortas were prepared for en face and elongation measurements as described previously.12 The incidence of ascending aortic ulceration was also quantified.

To quantify medial thickness and elastin breaks, aortas from each group were chosen based on measurement nearest to the mean of the arch area. Serial sections of ascending aortas were stained with Movat’s pentachrome.

Statistics

Analyses were performed by tests that were appropriate for the number of groups being compared and for the parametric or nonparametric characteristics of the data. Data are represented as means ± SEM. P<0.05 was considered statistically significant.

RESULTS

Whole body deficiency of AT1a receptors ablated ascending AAs

We first defined the effects of whole body AT1a receptor deficiency on AngII-induced ascending AA formation in LDL receptor −/− mice fed a diet enriched in saturated fat. Ascending aortic intimal area was measured as an index of severity of aneurysm. This area was similar in both AT1a receptor +/+ and −/− mice infused with saline. AngII infusion significantly increased the ascending aortic area in AT1a receptor +/+ mice (P<0.001). In contrast, there was no significant increase in this region of AT1a receptor deficient mice infused with AngII (Figure 1).

AT1a receptor deficiency in bone marrow-derived cells did not influence development of ascending AAs

Following the demonstration that whole body deficiency of AT1a receptors completely attenuated development of ascending AAs, we sought to determine the cell types expressing this receptor that were responsible for this pathology. To examine the role of hematopoietic cells, LDL receptor −/− mice that were either AT1a receptor +/+ or −/− were irradiated and repopulated with bone marrow-derived cells harvested from AT1a receptor +/+ or −/− donors. The development of ascending AAs was determined by the AT1a receptor genotype of recipients. AngII-infused into AT1a receptor +/+ recipients significantly increased arch area in both donor genotypes (Figure 2A), while AT1a receptor −/− recipients did not develop ascending AAs (Figure 2B). No significant effects were observed for donor genotype, irrespective of recipient AT1a receptor genotype.

AngII failed to contract SMCs in the ascending aorta

Since we failed to detect an effect of leukocytic AT1a receptors in forming ascending AAs, we next focused on mechanisms within the aortic wall. AngII infusion leads to changes localized to ascending aortic media layers.3 Therefore, we determined whether AngII exerted direct effects on SMC contractile responses that were differed in ascending aortas compared to other aortic regions. To accomplish this, we performed contractile studies using aortic rings from selected regions, as described previously.13 KCl and 5-HT contracted all aortic regions (ascending, descending thoracic, and infrarenal). However, AngII produced minimal discernible contraction of ascending aortas (Figure 3). Consistent with a previous report, only infrarenal aortic regions contracted in response to AngII stimulation.14 These studies failed to provide any evidence of differential contractile responses to AngII in SMCs of ascending and descending aortas.

AngII-induced aortic contractions were restricted to the infrarenal region

AT1a receptor deficiency on SMCs had no effect on development of ascending AAs

To determine whether AngII interacted directly with AT1a receptors on SMCs within ascending aortas to promote aneurysms, we generated mice with loxP sites on either side of exon 3 (Online Figure I). Mice were bred to transgenic mice expressing Cre under the control of an SMC promoter, SM22. Depletion of exon 3 of the AT1a receptor gene was confirmed by PCR using genomic DNA from aortas (Figure 4A). Depletion of AT1a receptor mRNA in SMCs was confirmed by RT-PCR (Figure 4B). As with whole body AT1a receptor deficiency,15 the deletion of AT1a receptors in SMC had no discernable effect of aortic structure.

SMC deficiency of AT1a receptor had no effect on AngII-induced ascending AAs

We attempted to determine the depletion of AT1a receptor protein. However, of the multiple commercial and custom developed antibodies, we were unable to demonstrate an interaction that was specific for the mouse AT1a receptor protein in either Western blotting or immunostaining of tissue sections. As an alternative approach to illustrate the uniformity of SM22-Cre-induced gene depletion across the aortic media, we bred SM22-Cre expressing mice to ROSA26 mice. ROSA26 mice have a suppressed β-galactosidase, but this enzyme is present in cells that expressed Cre at some point during lineage development. These studies demonstrated uniform β-galactosidase activity throughout the media of Cre transgenic mice, while there was no detectable activity in media extracted from non-transgenic littermates (Figure 4C).

Depletion of AT1a receptors in SMCs of LDL receptor −/− mice fed a high fat diet had no significant effect on body weight, serum cholesterol concentrations, or SBP (data not shown). Intimal areas of the aortic arch were similar in both groups. (Figure 4D.) These studies demonstrated that SMC-specific AT1a receptor deficiency had no effect on AngII-induced ascending AAs relative to littermate controls.

To determine the role of receptor expression on the endothelium, we bred AT1a receptor floxed mice to mice expressing Cre under the control of the Tek promoter. Depletion of the AT1a receptor from endothelial cells was confirmed by PCR using aortic endothelial cells cultured from Cre 0/0 and +/0 mice. (Figure 5A). Real time PCR confirmed deletion of AT1a receptor mRNA in endothelial cells (Figure 5B). In the absence of an AT1a receptor antibody to confirm depletion of the receptor protein, Tek Cre expressing mice were bred to ROSA26 mice to examine uniformity of AT1a receptor gene depletion of endothelial cells. There was uniform β-galactosidase activity throughout the endothelium of Cre +/0 mice, while there was no detectable activity in endothelium extracted from Cre 0/0 littermates (Figure 5C).

Depletion of AT1a receptors in endothelial cells had no significant effect on body weight, serum cholesterol concentrations, MCP-1 concentrations, or SBP (data not shown). However, deficiency in this cell type led to significant reductions in ascending aortic intima area expansion during AngII infusion (P<0.003; Figure 6A). The reduction was not complete and intimal area remained significantly increased over saline-infused mice (P=0.035). Using the length of the outer aortic curvature as an aneurysm index ascending aortas of endothelial cell-specific AT1a receptor deficient mice elongated less than in the wild type group during AngII infusion (P=0.014; Figure 6B). AngII infusion did not promote a significant increase in elongation in Cre +/0 mice, compared to saline infusion in this strain.

Tissue sections from the ascending aorta were stained with Movat’s pentachrome (Figure 7A). As described previously,6 these sections confirmed the increased media thickness with a major expansion of intra-elastin spaces being present in the adventitial aspect of the media. AngII infusion into Cre0/0 mice resulted in medial thickening with a blue-green stain that is indicative of glycosaminoglycan and proteoglycan deposition (Figure 7A,B). Tek-driven expression of Cre resulted in significant attenuation of AngII-induced expansion of medial thickness (P=0.01; Figure 7B). Elastin breaks with a random occurrence were present throughout the ascending aortic media following AngII-infusion. As with the medial thickness, this was significantly attenuated in the endothelial-specific Cre expressing mice relative to Cre 0/0 littermates (P=0.002; Figure 7C).

DISCUSSION

Numerous publications have demonstrated that AngII infusion into mice leads to development of AAAs.16,17 More recently, it has been observed that AngII infusion into mice also leads to pronounced aortic dilation and dissections that are highly restricted to the ascending aortic region.6,18 Although AngII generates aortic aneurysms in both regions, the pathology of ascending AAs demonstrates many distinctions, including concentric dilation without local medial rupture and medial thickening that is most pronounced on the adventitial aspect.6,19 This ascending aortic pathology that evolves during AngII infusion bears many similarities to a model of Marfan’s syndrome in which mice express a mutant allele of fibrillin-1.5

In the current study, whole body deficiency of AT1a receptors ablated development of AngII-induced ascending AAs. To define the cell type expressing AT1a receptors that contributed to development of ascending AAs, a combination of approaches was taken using bone marrow transplantation and cell-specific conditional deficiencies. These approaches studied three major cell types in AngII-induced ascending AAs: leukocytes, SMCs, and endothelial cells. Using these approaches, we were unable to detect any effect of AT1a receptor deficiency in leukocytes or SMCs on development of AngII-induced ascending AAs. In contrast, depletion of AT1a receptors in endothelium attenuated development of AngII-induced ascending AAs.

Many of the physiological and pathological effects of AngII are mediated by stimulation of AT1 receptors.20 In rodents, chromosomal duplication has led to expression of two subtypes of AT1 receptors, termed a and b.21 Although AT1b receptor expression is more restricted compared to AT1a receptors, aortic tissue contains both subtypes with a predominance of the b subtype.22,23 Only the b subtype is involved in AngII-induced contractile activity that has been previously demonstrated to be restricted to the infrarenal region.24,25 Despite the presence of functional AT1b receptors in aorta, AT1a receptors plays an essential role in AngII-induced ascending AAs based on the ablation of the pathology in AT1a receptor deficient mice. It may be presumed that minor amino acid differences between AT1a and AT1b receptor subtypes lead to differential stimulation of signaling pathways that differentiate AngII-induced contractile versus pathological processes.26

Leukocytes are prominent components in both AngII-induced AAAs and ascending AAs.6,19 Evaluation for a role of AngII acting directly on leukocytes was performed in AAAs using bone marrow transplantation to create mice that are chimeric for AT1a receptors.8 While this approach did not distinguish the cells types that arise from the bone marrow, macrophages are the most dominant cell type from this origin in AngII-induced AAAs.18,19,27 Similarly, macrophages are the most abundant leukocytes in AngII-induced ascending AAs. Repopulation of irradiated mice demonstrated no effect of AT1a receptors on donor cells to development of AngII-induced AAAs.8 The current study also failed to demonstrate a role for AT1a receptors on donor cells in promoting AngII-induced ascending AAs. Furthermore, while functional AT1 receptors have been identified on mouse macrophages,28 we were unable to demonstrate a role for AT1a receptors on this cell type in directly influencing development of ascending AAs.

AngII is known to exert region-specific effects on aortic SMCs in physiological and pathological responses.14,29 We have demonstrated previously that the mechanism of AngII-induced SMC-rich medial thickening in vivo differs in the ascending aorta relative to all other regions.15 This difference may be a consequence of the different developmental origin of SMCs throughout the aortic tree.30 In the present study, we initially sought to demonstrate whether any regional differences in SMC responses to AngII can be detected ex vivo. Previous studies have demonstrated that contractile responses to AngII differ between aortic regions, with minimal contraction in the descending thoracic aorta compared to the infra-renal region.14,25 Responses may differ in the ascending aorta since SMCs in this region have a different developmental origin from the descending aorta, with the potential for different functional characteristics.31,32 However, similar to the descending aorta, we were also unable to detect any AngII-induced SMC contraction in rings isolated from the ascending aorta, despite this region contracting to KCl and 5-HT. Therefore, AngII does not have demonstrable effects on a physiological function of SMCs in this aortic area.

To directly determine the role of AngII stimulation of SMCs in the pathological process of ascending AAs development, we generated AT1a receptor floxed mice to develop cell-specific deficient mice. The floxed sites were located either side of exon 3 that is responsible for the entire translatable portion of the protein. AT1a receptor deficiency on SMCs was created by breeding mice that were hemizygous for Cre driven by the SM22 promoter.33 This Cre expression strategy has been used previously to determine the role of SMCs in experimental AAAs, but has not been used to determine ascending AAs.33–35 The normal mouse aorta not only has different developmental origins of SMCs, but the media may also contain other cell types, such as fibroblasts and myofibroblasts.36 Despite this potential heterogeneity, studies in which SM22 driven Cre was expressed in ROSA26 mice demonstrated the uniformity of gene depletion across the media that coincided with α-actin immunostaining. Although we were unable to validate spatial distribution of AT1a receptor protein expression, the combined evidence from the SM22-Cre-ROSA26 mice and the absence of AT1a receptor mRNA in aortic SMCs is consistent with an effective depletion. In mice with this depletion, we were unable to detect any difference in AngII-induced ascending AAs. This is consistent with the lack of direct effects of AngII on SMCs to initiate and propagate AAs.

Endothelial function has a profound effect on vascular physiology and pathology and endothelial cells express angiotensin II receptors.37 To determine a role of AngII stimulation on endothelial cells in the development of AngII-induced ascending AAs, we bred AT1a receptor floxed mice to those expressing Cre under the control of the Tek promoter. Use of the Tek promoter has the potential complication of deleting genes in cells of myeloid origin, in addition to endothelial. Given that myeloid and endothelial cells are derived from a common precursor cell of the hemangioblast, any genes expressed at this stage of development would have a broader spectrum of effects on these different cell types.38 However, since we were unable to demonstrate any effect of AT1a receptor expression on bone marrow-derived cells, the attenuation of AngII-induced ascending AAs in Tek-driven Cre mice was not confounded by deletion of the receptor in myeloid cells.

Although deficiency of AT1a receptors in endothelium attenuated the development of AngII-induced ascending AAs, the effect was not as profound as the complete ablation of disease in mice with whole body AT1a receptor deficiency. In contrast, deficiency of AT1a receptors in SMCs had no discernible effect on the development of aneurysms. Furthermore, bone marrow transplantation studies failed to reveal any effect of leukocyte AT1a receptor deficiency on aneurysm development. Therefore, of the three major cell types present in aneurysmal tissue, only depletion of AT1a receptors in the endothelium reduced aneurysmal disease, but not as dramatically as whole body deficiency. One potential explanation is a technical shortcoming that the activity of Cre was not sufficient to delete the floxed exon 3. It would have been preferable to demonstrate deletion by determination of changes in AT1a receptor protein. Unfortunately, we were unable to validate that commercially available antibodies authentically stained the AT1a receptor protein. We were also unable to develop antibodies that specifically recognize this protein. Therefore, the index of deletion was dependent on measurement of mRNA abundance. In agreement with previous studies using Cre under the control of SM22 Cre,33,34,39 we demonstrated a highly effective deletion of the floxed components of the gene. Given the inability to isolate mRNA from the endothelial cells harvested from the ascending aorta, we performed this analysis on endothelial cells isolated from lung. Findings in these cells were directly complemented by studies using Tek expressing mice in the ROSA26 background. Analyses of sections from ascending aortas of these mice demonstrated a uniform expression of β-galactosidase in the endothelium. Therefore, expression of Cre in either endothelial or smooth muscle selective manner appears to have been effective in removing the exon that translates the entire functional protein.

Another explanation of the different extent of whole body versus cell-specific AT1a receptor deficiency on ascending AAs may be the involvement of other cell types. There is emerging evidence for a role of fibroblasts and myofibroblasts in the development of experimental aneurysms by exposure to calcium chloride or AngII infusion.18,36,40 These cell types have been detected in both the media and adventitia of mice. Future studies in mice expressing Cre under a fibroblast-specific promoter will provide insight into the role of this cell type.

Depletion of AT1a receptors in endothelial cells reduced the development of AngII-induced ascending AAs to a similar extent to that previously described in mice deficient in CCR2, the cognate receptor for monocyte chemoattractant protein-1 (MCP-1). With both deficiencies there was a decrease in lumen area and medial thickness. AngII can stimulate the release of MCP-1 from endothelial cells.41 Therefore, it is interesting to speculate that AngII stimulation of endothelial AT1a receptor promotes MCP-1 release that permeates the media due to blood pressure. This could promote leukocyte recruitment via the adventitial aspect of the aorta with the subsequent elaboration of elastolytic enzymes. The recent availability of MCP-1 floxed mice will permit the testing of this hypothesis.42

In summary, we demonstrate that endothelial-specific deficiency of AT1a receptors markedly attenuates the development of AngII-induced AAs. Future studies will define the nature of the mediator released from the endothelium that promotes this localized disease.